MITF (an abbreviation for microphthalmia-associated transcription factor) is one of transcription regulators present in organisms; it is a protein capable of modulating an expression of a c-kit gene, which is specific to mast cells.
MITF is a known substance (Cell, vol. 74, 395404, 1993); it was, however, a gene that codes for MITF that was first discovered. That is to say, the gene was isolated as a causal gene for a mi/ml mouse. In the mi/ml mouse, because of a mutation in the MITF gene, that is, a deletion of one amino acid in the transcription activation region of the MITF gene, normal MITF is not expressed. The mi/ml mouse is a mutant mouse having hypoplasia of an eyeball, melanocyte deficiency, mast cell loss, and osteopetrosis of a bone as main symptoms thereof, and presents differentiation anomalies in tissues, such as melanocytes, mast cells, retinal pigment epithelial cells, and osteoclasts. These cell differentiation disorders in the mi/ml mouse are attributed to the fact that normal MITF is not expressed, and gene transcription is not activated by MITF.
Furthermore, in Northern-blot analyses of a tissue distribution of an expression, using tissues or cell lines from a normal mouse, it was found that MITF mRNA was expressed in the heart, in melanocytes, and in mast cells. In recent years, the existence of MITF isoforms has been reported; i.e. a melanocyte type (M type), a heart type (H type), and an A-type, in which the cDNA sequences were different at the 5′ end [Seikagaku (Journal of the Japanese Biochemical Society), vol. 71, no. 1, 61–64, 1999]. The MITF gene comprises 10 (M type) or 11 (A and H types) exons, and all three types are substantially common beyond exon 2. In the M type, two further types are distinguished by an addition, or lack thereof, of exon 5b, which comprises 18 bases coding for 6 amino acids. The A and H types completely match each other beyond exon 1B, exon 1 at the 5′ end being different. The A and H types and the M type are common beyond exon 2; however the M type has no exon 1B upstream of exon 2, which is linked to a specific exon 1. Furthermore, it has been shown that the promoter for each type is different from the genomic sequence.
An MITF protein is assumed to comprise a nuclear localization region, a transcription activation region, a DNA binding region, a dimerization region, and an activation region for MITF itself; the presence of these regions is common to all known types. The MITF protein is a transcription regulator, which has a bHLH-Zip (base-helix-loop-helix/leucine zipper) motif at a center of its structure, it forms a dimer to bind to DNA, and activates transcription of targeted genes. Since major differences have been reported between transcription activation capabilities of the A-type and the H type, there is assumed to be a function that regulates the transcriptional activity at exon 1, which differs in genetic sequence between the two types.
Furthermore, it has been reported that the MITF protein works as a transcription regulator in melanocytes, and that it is involved in the multiplication and differentiation of melanocytes, as well as in the melanin synthesis pathway, and the like.
It has been reported that the MITF protein also acts as a transcription regulator in mast cells, modulating the expression of the c-kit gene (a transcription factor that activates the c-kit promoter) (Blood, vol. 88, no. 4, 1225–33, 1996). The c-kit gene is expressed in hematopoietic precursor cells, mast cells, pigment cells, and germ cells and regulates the multiplication and the differentiation of these cells by the action of the Si factor. It is thought that, in mast cells, MITF protein is involved in mast cell survival maintenance by regulating the expression of c-kit gene expression.
Mast cells have long been reported to be involved in allergic diseases [“IgE, Mast Cells and the Allergic Response” (Ciba Foundation symposium) 147, John Wiley & Sons, Chichester, UK, 1989]. Furthermore, diseases other than allergic diseases in which mast cells are involved include autoimmune diseases, pulmonary fibrosis, carcinomas, mastocytosis, mastocytoma, and the like.
Meanwhile, PTD (an abbreviation for Protein Transduction Domain) is a general term for domains to penetrate the biological membrane and transfer proteins into the cells (uptake). For example, in analysis of HIV antigens by domain units, it is confirmed that a TAT-derived peptide portion serves to transfer the HIV antigen inside normal T-cells, which is one of contributing factors in cell infection (Cell, vol. 55, 1179–88, 1988). Based on such observations, there have been reports of the existence of various PTDs to work in a similar way to TAT, and of techniques whereby these PTDs are fused with various proteins for translocation into cells (Current Opinion in Molecular Therapeutics 2000, vol. 2, no. 2, 162–67, 2000).
However, there have been no reports to date of techniques related to MITF or mast cells to transfer into cells using PTD.